Method and reagents for inactivating ribonucleases RNase A, RNase I and RNase T1

ABSTRACT

The present invention is a general method for irreversibly inactivating ribonucleases. Ribonucleases are completely inactivated by treating them with a reducing agent and heat. RNA samples contaminated with ribonuclease may be treated with this method to protect them from degradation. The RNA may then be used directly in a variety of enzymatic reactions and molecular biology techniques. This method may also be applied to a variety of molecular biology reagents which may be contaminated with ribonuclease to protect an RNA from being degraded when incubated with the reagent.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology. More particularly, it concerns the inactivation ofribonucleases (RNases) which can degrade RNA.

2. Description of Related Art

The quality of an RNA preparation greatly affects the results obtainedwhen analyzing it by a number of different molecular biology techniquessuch as northern blotting, ribonuclease protection assays and RT-PCR(Reverse Transcriptase-Polymerase Chain Reaction). Degraded RNA willproduce a lower signal than in an equivalent intact RNA sample.

RNA is much more susceptible to degradation than DNA (Sambrook et al.,1989). RNA is readily hydrolyzed when exposed to conditions of high pH,metal cations, high temperatures and contaminating ribonucleases. Amajor cause of RNA degradation is ribonuclease contamination, and thismust be guarded against in virtually all RNA-related procedures,including RNA isolation, mRNA purification, RNA storage, northernblotting, nuclease protection assays, RT-PCR, in vitro transcriptionand/or translation and RNA diagnostics. In addition to the endogenousribonucleases from cells and tissues, finger grease and bacteria and/orfungi in airborne dust particles are common sources of ribonuclease. Tominimize ribonuclease contamination, appropriate precautions must befollowed when handling RNA (Blumberg, 1987; Wu, 1997).

Ribonucleases are difficult to inactivate. For example, while at 90° C.,bovine pancreatic ribonuclease A (RNase A) has no activity. However, ifthe enzyme is quickly cooled to 25° C., the activity is fully restored.This process is known as reversible thermal denaturation. If the RNase Ais incubated at 90° C. over time, then decreasing fractions of theactivity are recovered at 25° C. This process is known as irreversiblethermoinactivation. At 90° C., it takes several hours to inactivateRNase A (Zale and Klibanov, 1986). Much of the lost activity isattributed to disulfide interchange (Zale and Klibanov, 1986). Further,the inventors and others have found that ribonucleases can evenwithstand autoclaving (121° C., 15 psi, 15 minutes) to some degree.Spackman et al. (1960) tested the stability of RNase A and concludedthat it was stable to heat, extremes of pH, and the protein denaturant,urea, results emphasizing the difficulty researchers have hadinactivating ribonucleases. For the above reason, a variety of methodsother than heating have been developed to inhibit or inactivateribonucleases. These methods, and their disadvantages, are describedbelow.

In one method of destroying RNases, diethyl pyrocarbonate (DEPC) isadded to final concentration of 0.1% to molecular biology reagents,glassware or electrophoresis apparatus, followed by incubating at 37° C.for several hours and then autoclaving for 15-20 minutes to destroy theDEPC (Wolf et al., 1970). DEPC reacts with the ε-amino groups of lysineand the carboxylic groups of aspartate and glutamate both intra- andintermolecularly (Wolf et al., 1970). This chemical reaction formspolymers of the ribonuclease. However, there are several disadvantagesto using DEPC: (1) It is a possible carcinogen and is hazardous tohumans; (2) some commonly used molecular biology reagents such as Trisreact with and inactivate DEPC; (3) treatment of samples with DEPC istime-consuming; (4) DEPC reacts with the adenine residues of RNA,rendering it inactive in in vitro translation reactions (Blumberg, 1987)and 5) If all of the DEPC is not destroyed by autoclaving, remainingtrace amounts may inhibit subsequent enzymatic reactions.

Traditionally, RNA is stored in DEPC-treated water or TE buffer.However, the RNA is not protected from degradation if the sample or thestorage solution has a minor ribonuclease contamination. It has beensuggested that RNA be stored in ethanol or formamide to protect an RNAsample from degradation because these environments minimize ribonucleaseactivity (Chomczynski, 1992). The obvious disadvantage is that the RNAsample cannot be directly utilized for analysis or enzymatic reactionsunless the ethanol or formamide is removed.

Guanidinium thiocyanate is commonly used to inhibit RNases during RNAisolation (Chomczynski and Sacchi, 1987; Sambrook et al., 1989). A highconcentration of guanidinium thiocyanate combined with β-mercaptoethanolis used to isolate RNA from tissues, even those that are rich inribonucleases, such as pancreas (Chirgwin et al., 1979). Guanidinium isan effective inhibitor of most enzymes due to its chaotropic nature.However, if RNA is dissolved in guanidinium, then it must first bepurified from the guanidinium prior to being used in an enzymaticreaction.

Vanadyl-ribonucleoside complexes (VRC) may be used to inhibit RNasesduring RNA preparation (Berger and Birkenmeier, 1979). The drawback tousing VRC, is that VRC strongly inhibits the translation of mRNA incell-free systems and must be removed from RNA samples by phenolextraction (Sambrook et al., 1989).

Favaloro et al. (1980) employed macaloid, a clay, to absorb RNases. Alimitation of this method is that it is difficult to completely removethe clay from RNA samples. Other reagents have been used to inhibitribonucleases including SDS, EDTA, proteinase K, heparin,hydroxylamine-oxygen-cupric ion, bentonite and ammonium sulfate(Allewell and Sama, 1974; Jocoli and Ronald, 1973; Lin, 1972; Jones,1976; Mendelsohn and Young, 1978). None of these reagents are stronginhibitors alone, although their inhibitory effect may be improved byusing them in combination. Like many of the RNase inhibitors alreadydescribed, although these chemicals inhibit RNase activity, they alsomay inhibit other enzymes such as reverse transcriptase and DNase I.Therefore, the RNA must be purified away from the inhibitory reagent(s)before it can be subjected to other enzymatic processes.

Two types of proteinaceous RNase inhibitors are commercially available:human placental ribonuclease inhibitor (Blackburn et al., 1977) andPRIME Inhibitor™ (Murphy et al., 1995). RNases of the class A familybind tightly to these protein inhibitors and form noncovalent complexesthat are enzymatically inactive. The major disadvantage of theseinhibitors is that they have a narrow spectrum of specificity. They donot inhibit other classes of RNases. Another disadvantage when usingplacental ribonuclease inhibitor is that it denatures within hours at37° C., releasing the bound ribonuclease. Thus, the RNA sample is onlyprotected for only a few hours at most.

Reducing agents are frequently used as adjuvants to RNA isolationsolutions in conjunction with denaturants to reduce the disulfide bondsin RNases that are rendered accessible by the denaturant. Commonly usedreducing reagents are β-mercaptoethanol, dithiothreitol (DTT),dithioerythritol (DTE), and glutathione. Another such reducing agent isthe amino acid cysteine. β-mercaptoethanol is often included in RNAisolation solutions combined with guanidinium thiocyanate to reduceribonuclease activity and solubolize proteins (Chomcyznski and Sacchi,1987). DTT is the strongest reducing reagent of the three listed.

DTT has low redox potential (−0.33 volts at pH 7.0) and is capable ofmaintaining monothiols effectively in the reduced form and of reducingdisulfides quantitatively (Cleland, 1964). DTT acts as a protectiveagent for free sulfhydryl groups. It is highly water soluble, withlittle tendency to be oxidized directly by air, and is superior to otherthiols used as protective reagents. DTT's reducing activity can beaccurately assayed using 5, 5′-dithiobis (2-nitrobenzoic acid) or DTNB(Cleland, 1964). The reduction of DTNB mediated by DTT generates ayellow color whose absorbance can be measured at 412 nm using aspectrophotometer. RNase A, RNase 1 and RNase T1 all contain disulfidebonds (Ryle and Anfinesen, 1957; Barnard, 1969) and, therefore, aresusceptible to reduction.

DTT has been used as an inhibitor of RNase A in the isolation ofpolyribosomes (Boshes, 1970; Aliaga, 1975). In Boshes' experiment,polyribosome preparations were treated with RNase A (10 μg/ml) insolution A (10 mM MgCl₂: 10 mM Tris [pH 7.6 ]: 50 mM KCl) in thepresence or absence of 4 mM DTT at 4° C. for 20 minutes. The treatmentof polyribosomes with RNase A generated monoribosomes. Boshes observedthat polyribosomes treated with RNase A in the presence of 4 mM DTTreduced the conversion of polyribosomes to monoribosomes and from thatresult he concluded that DTT was an RNase inhibitor. Boshes' statementwas based on the effect of DTT on the conversion of polyribosomes tomonoribosomes by RNase A. He did not directly assay the degradation ofpurified RNA. Since Boshes was working with a complex, uncharacterizedprotein mixture, it is unclear as to what may have been responsible forthe decreased production of monoribosomes. For example, the addition ofDTT may have increased the activity of the endogenous mammalian RNaseinhibitors rather than act directly on the RNases. These RNaseinhibitors require a reducing environment for activity.

Heat has been used to inactivate RNase A by mediating the breakage ofdisulfide bonds. Zale and Klibanov (1986) performed inactivation ofRNase A at 90° C. and pH 6.0 for 1 hour, which induced the followingchemical changes: disulfide interchange, β-elimination of cysteineresidues, and deamidation of asparagine. This type of heat treatment didnot completely inactivate the ribonuclease. A major disadvantage is thata long-term, high-temperature treatment (90-100° C.) is incompatiblewith RNA. Such treatment promotes the hydrolysis of RNA. In fact, theinventors have found that total RNA incubated at 65° C. for severalhours is almost completely degraded. Thus, treating an RNase sample withextreme heat to inactivate ribonucleases will mediate the distruction ofthe RNA which the user is trying to protect.

SUMMARY OF THE INVENTION

The present invention provides a general method for rapidly inactivatingribonucleases and RNA storage solutions adapted for use in such methods.These methods comprise the steps of obtaining a sample; obtaining areducing agent; admixing the reducing reagent and sample; and heating.

The inventors' method for inactivating ribonuclease can protect RNA fromribonuclease degradation during storage. More importantly, the RNAsamples can be used immediately after ribonuclease inactivation for RNAanalysis, and in enzymatic reactions such as the synthesis of cDNA byreverse transcriptase and the degradation cellular DNA by DNase I.

As used herein, the terms “RNase inactivation” or the “inactivation ofRNases” denotes that there is no detectable degradation of the sampleRNA under the assay conditions used.

In one embodiment, the present invention relates to reagents for use inmethods for inactivating ribonucleases and such methods comprising: (a)obtaining a sample; (b) obtaining a reducing agent; (c) admixing thesample and the reducing agent; and (d) heating the admixture, whereinribonucleases in the sample are inactivated. There are many differentmanners in which the methods and reagents of the present invention maybe used. However, in a preferred embodiment, the reagents and methodswill be used to inactivate any ribonucleases that are present in asample that is a reagent used in molecular biology, either in caseswhere ribonuclease contamination is known or expected to have occurred,or simply as an additional prophylactic step in attempts to avoid RNasecontamination. As discussed above, ribonuclease contamination canseriously affect the results of molecular biological assays. Therefore,there is great value in having simple, efficient methods for preventingribonuclease contamination of molecular biology reagents. In many cases,the molecular biology reagent will be one employed in the handling ofRNA, water, TE buffer, 20×SSC, 10×MOPS, Tris buffer, EDTA, nucleic acidhybridization buffer, sodium acetate buffer, formalin tissue fixative,in situ hybridization buffer, or nucleic acid storage buffer/solution.

Any agents and reagents having reducing properties are well known tothose of skill in the art, and, by employing assays described herein,one of ordinary skill in the art will be able to determine which of anyof these reducing agents will be of use in the present invention.Presently preferred reducing agents are those comprising DTT,β-mercaptoethanol, cysteine, or dithioerithritol. The finalconcentration of these reducing agents in the solution that is to beprotected from ribonuclease can be any which functions to achieve theribonuclease protective activity that is the purpose of the invention.Presently preferred concentrations are those between 0.5 and 500 mMreducing agent in the admixture of the sample and the reducing agent.More presently preferred concentrations are between 1 and 200 mM in theadmixture. Even more presently preferred concentrations are between 2and 60 mM. The presently most preferred concentration is 20 mM in theadmixture. Of course, the invention is in no way limited to thesepreferred concentrations.

In most cases, the reducing agent is comprised in a buffer solutionprior to admixing. The buffer solution will comprise the agent at a highenough concentration such that the final concentration of the agent inthe admixture is sufficient to realize the ribonuclease protective goalsof the invention. The buffer solution may also comprise a chelator suchas sodium citrate, EGTA, or EDTA.

The admixture may be heated to any temperature and for any amount oftime that is sufficient to accomplish the ribonuclease inactivationgoals of the invention. Presently preferred conditions call of theheating of the admixture to at least 37° C. for at least 4 minutes Theheating may be accomplished by any means standardly employed in abiological lab. The inventors typically use a heat block or water baththat has been set to the desired temperature.

One of the advantages of the present invention is that the methods donot affect the stability of any RNA that is comprised in the solutionthat is being treated to inactivate ribonucleases. Therefore, a specificembodiment of the invention comprises methods for inactivatingribonucleases in the presence of RNA comprising: (a) obtaining an RNAsample; (b) obtaining a reducing agent; (c) admixing the sample and thereducing agent; and (d) heating the admixture, wherein ribonucleases inthe sample are inactivated. The RNA sample may be any sample thatcontains RNA, including but not limited to a tissue sample, a cellsample, a crude cell preparation, isolated total RNA, and any form ofpurified RNA. In a preferred embodiment, the sample is comprised ofpurified RNA. The reducing agents, other components, concentrations,times, and temperatures are typically as described above.

In some embodiments, the invention relates to methods for sequentialinactivation of any ribonucleases in a sample such that anyribonucleases introduced to the sample at some time after a firstinactivation procedure may be inactivated comprising: a) obtaining asample; b) obtaining a reducing agent; c) admixing the sample and thereducing agent; d) performing a first heating of the admixture, wherebyany ribonucleases in the admixture are inactivated; e) determining thata further inactivation procedure is warranted to inactivate anyribonucleases that may have been introduced to the sample subsequent tothe first heating; f) performing a second heating of the admixture,whereby ribonucleases in the admixture are inactivated. The benefits ofthese methods are clear. One may have a sample of RNA that also containsan appropriate amount of reducing agent. Prior to storage of the RNA,one can simply heat the sample for an appropriate length of time to anappropriate temperature, thereby inactivating any ribonucleases. Thenext time the RNA is accessed, the remaining RNA may be reheated toinactivate any ribonucleases that might have been introduced into thesample by the access procedure. This process may be carried out anindefinite number of times throughout the storage life of the RNA.

The present invention also comprises solutions for storing RNAcomprising: a) a reducing agent; and b) an RNA sample. In some preferredembodiments, the reducing agent will be selected from the groupconsisting of DTT, dithioeritol, β-mercaptoethatnol, and cysteine. Thefinal concentration of the reducing agent in the solution can be anywhich functions to achieve the ribonuclease protective activity that isthe purpose of the invention upon heating. Presently preferredconcentration are those between 0.5 and 500 mM reducing agent in thesolution. More presently preferred concentrations are between 1 and 200mM in the solution. Even more presently preferred concentrations arebetween 2 and 60 mM. The presently most preferred concentration is 20 mMin the solution. Of course, the invention is in no way limited to thesepreferred concentrations. Some preferred RNA storage solution comprise abuffer, and presently preferred pHs of the solution are between 5.0-7.0.High pHs mediate the hydrolysis of RNA. However, any pH that does notadversely affect the RNA may be used. The solution may also comprise ametal chelator such as, for example, sodium citrate, EGTA, or EDTA. Thechelator is sometimes desired because some metal cations can mediate thehydrolysis of RNA in a sample.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. DTT-heat inactivation of different RNase concentrations. TheRNase mixture was made to different concentrations in 20 mM DTT: 1 mMsodium citrate (pH 6.4). The RNase samples were incubated at 60° C. for20 minutes. A radioactive RNA probe was added to the treated RNasesamples, incubated at 37° C. for 16 hours, fractionated in a denaturing5% acrylamide gel and exposed to x-ray film. The (+) lane is the RNAprobe which did not contain DTT or RNase but was incubated at 37° C. for16 hours.

FIG. 2. DTT does not inhibit the AMV or MMLV reverse transcriptases.Reverse transcriptions were performed using the RETROscript™ kit(Ambion, Inc.). Mouse total RNA in DEPC-treated water was reversetranscribed with either MMLV or AMV reverse transcriptase in thepresence or absence of 20 mM DTT. dCTP[α³²P] was included in thereactions to measure the efficiency of reverse transcription. Followingincubation at 42° C. for 1 h, 5 μl of each reaction was subjected to TCAprecipitation to measure the amount of dCTP[α³²P] incorporated into cDNAby reverse transcription.

FIG. 3. DTT does not inhibit T7, T3 and SP6 RNA polymerases.Transcriptions were performed with T7, T3 and SP6 RNA polymerase in thepresence and absence of 20 mM DTT. For each RNA polymerase reaction,pTRI-Xef DNA was the template and UTP[α³²P] was included to measure theefficiency of transcription. Duplicates were performed for eachreaction. TCA precipitations were performed on each transcriptionreaction to measure the percent of the UTP[α³²P] incorporated into RNA.

FIG. 4. Time dependence of RNase inactivation. The RNase mixture wasmade to 200 ng/ml in 1 mM sodium citrate (pH 6.4) in the presence of 20mM DTT. 5 μl aliquots of the RNase mixture was heated for increasingamounts of time at 60° C. After heating, 4 μl (1 μg/μl) of total mousebrain RNA was added to each of the samples and incubated at 37° C. for 1h. The samples were then fractionated on a formaldehyde denaturing 1%agarose gel in the presence of ethidium bromide. The RNA was detected byUV fluorescence. The lane labeled with the (+) is the mouse RNAsubstrate which was not subjected to any RNase. The lane labeled withthe (−) is the mouse RNA substrate which was subjected to the RNase and20 mM DTT without any heating.

FIG. 5. DTT is oxidized by incubating at 65° C. for 6 days. DTT was madeto 100 mM in water and incubated at 65° C. for 6 days. Fresh DTT and theheated 100 mM were diluted in water to different concentrations andsubjected to the DTNB test. Absorbance at 412 nm indicates the reducingstrength of the DTT.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to methods for inactivating ribonucleases,which employ at least one reducing reagent and heat of at least 37° C.Generally, the reducing agent is added to buffers, solutions or watercontaining a potential RNase contamination. RNases are then inactivatedby heating. The preferred reducing reagents are DTT, DTE, cysteine, andβ-mercaptoethanol. To effectively inactivate RNases, the presentlypreferred method is to add DTT to a final concentration of 20 mM to thesample and then incubate at 60° C. for 8 minutes. The following examplesprovide detailed description and utilities of the present invention.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Criteria for the Analysis of Ribonuclease Inactivation

The inventors routinely perform assays on RNA designed to assess RNaseactivity in a sample. Two different assays are typically used for thedetection of ribonuclease activity, non-isotopic and isotopic. Eachassay generates similar data with regard to the sensitivity ofdetection. In both assays, the inactivation process is typicallyperformed on a mixture of three different ribonucleases: RNase A, RNase1, and RNase T1. Each ribonuclease is purified from a different species:human, E. coli and a fungus, respectively. The three RNases are verydifferent from each other in their origin, substrate specificity andprotein sequence. In this way, the inactivation process can test threecompletely different but well characterized ribonucleases. The standardstock RNase mixture employed in the following examples is prepared bycombining 1 μl RNase 1 (100 U/μl; Ambion), 1 μl RNase cocktail (RNase A,0.5 U/μl; RNase T1, 20 U/μl, Ambion) and 98 μl water.

By employing one or both of these assays, one of skill will be able todetermine additional reducing agents that function in the invention. Inorder to do so, one need only obtain a putative reducing agent that isexpected to have RNase inactivating activity and then perform the typesof assays performed herein with regard to DTT, cysteine, etc. todetermine the utility of the putative reducing agent in the methods andcompositions of the invention.

1. Non-Isotopic RNase Inactivation Assay

The non-isotopic assay uses total RNA isolated from mouse as thesubstrate for the RNase mixture. The assays reported in the followingexamples were typically performed in a final volume of 10 μl. A quantityof4 μg of total RNA isolated from mouse liver or brain was dissolved inan aqueous solution, usually water or 1 mM sodium citrate (pH 6.6). Thetreated ribonuclease sample was added to the total RNA and thenincubated at 37° C. for 1 or 16 hours, depending on the sensitivitydesired for the assay. After incubation, the RNA was fractionated in aformaldehyde 1% agarose gel. The RNA was detected by staining withethidium bromide and then illuminating the gel with ultraviolet light.The RNA fluoresces in the gel. Untreated total RNA was also fractionatedas a control with the test samples for comparative purposes. Testsamples containing inactivated RNase produced the same ethidium bromidestaining pattern as the untreated RNA control. Intact total RNA has twomajor bands produced by the 28S and 18S ribosomal RNA. If theintensities of the ribosomal RNAs became diminished compared to thecontrol RNA, then the RNases were not inactivated by the inactivationtreatment. An example of the non-isotopic assay in use in found inExample 6 and the Figure cited therein.

2. Isotopic RNase Inactivation Assay

The isotopic RNase assay uses a radioactive RNA synthesized by in vitrotranscription as the RNA substrate. The RNA used in the followingexamples was 1.9 or 4.0 kb in length. The radioactive RNA wassynthesized using a T7 MEGAscript™ transcription kit (Ambion, Inc.). Thein vitro transcription reaction mixture contained 1.0 μg of linearizedDNA template, 2 μl of 10×transcription buffer, 1 μl of UTP[α³²P] (800Ci/mmol), 2 μl of each 75 mM ribonucleotide and 2 μl of the T7 RNApolymerase mix, final volume of 20 μl. The reaction was incubated at 37°C. for 3 hours. The transcript was precipitated with 7.5 M LiCl andresuspended in 0.1 ml of 1 mM sodium citrate (pH 6.6) or RNase freewater.

2 μl of the RNA probe was incubated with the test sample in a finalvolume of 10 μl for about 16 hours at 37° C. After incubation, the RNAwas fractionated in a denaturing 6 M urea 5% acrylamide gel and then thegel was exposed to x-ray film. Untreated RNA was also fractionated as acontrol with the test samples for comparative purposes. Test samplescontaining no detectable RNase activity produced the same single band asthe untreated control RNA. RNase activity was indicated by the intensityof the RNA decreasing and by the appearance of smearing below the intactRNA. An example of the isotopic assay in use is found in Example 3 andthe Figure cited therein.

EXAMPLE 2 Boshes' Method does not Inactivate RNases

Boshes (1970) stated that DTT is an RNase inhibitor, a statement basedon the observation that the addition of DTT decreased the generation, byRNase A, of polyribosomes to monoribosomes in a crude polyribosomepreparation. RNase A (10 μg/ml) activity was inhibited by the additionof 4 mM DTT at 4° C. for 20 minutes (Boshes, 1970). The inventorsdemonstrated that these conditions are insufficient to inactivate RNaseusing their assay system. RNase A, at 200 ng/ml instead of 10 μg/ml, wastreated with or without 4 mM DTT in Boshes' solution A at 4° C. for 20minutes. RNase A activity was then tested by incubating mouse livertotal RNA with the treated RNase A at 37° C. for 60 minutes. The mouseRNA was then fractionated by electrophoresis in a 1% agaroseformaldehyde gel. RNase A at 200 ng/ml was not inactivated using theconditions set forth by Boshes (1970). In fact, the RNA was completelydigested. In contrast, the RNase A treated at 60° C. for 20 minutes inthe presence of 20 mM DTT was completely inactivated, i.e., the mousetotal RNA remained completely intact.

A likely explanation for the observed results is that Boshes used acrude, uncharacterized sample containing a complex set of proteinsassociated with the polyribosomal fraction. Mammalian RNase inhibitor isfound in many tissues and is dependent on reducing conditions foractivity. RNase inhibitor binds to RNase to inhibit its activity. Underoxidizing conditions, the RNase inhibitor releases active RNase. Thuswhen Boshes added DTT to his preparation, he may have restored orincreased the activity of the naturally occurring RNase inhibitor.

EXAMPLE 3 Use of Reducing Agents Coupled with Heat InactivateRibonucleases

1. Concentrations of RNase Mixture Inactivated by Treating with aReducing Reagent and Heat.

The RNase mixture was added to 1 mM sodium citrate (pH 6.4) containing20 mM DTT up to final concentration of 1 μg/ml and incubated at 60° C.for 20 minutes RNase activity was tested using the non-isotopic (1 hourincubation) and the isotopic assays (FIG. 1). As measured by bothassays, the RNase mixture was completely inactivated up to 200 ng/ml.

In related experiments, β-mercaptoethanol and DTE at 20 mM wereincubated with the RNase mixture at 200 ng/ml at 60° C. for 20 minutes.These reducing reagents had the same effect as DTT in that they wereable to inactivate RNase activity completely as measured using thenon-isotopic assay.

2. Inactivating RNases in the Presence of RNA

The RNase mixture was added at final concentrations of 200, 100, 50, 25,12.5, 6.25, 3.125, 1.56, 0.78 and 0.39 ng/ml to mouse liver total RNA (4μg/10 μl) in 1 mM sodium citrate (pH 6.4): 20 mM DTT. The samples wereincubated at 60° C. for 20 minutes and then assessed for RNase activityusing the non-isotopic assay, incubating 37° C. for 1 hour. This methodof RNase inactivation was able to completely inactivate the RNasemixture up to 3.125 ng/ml in the presence of RNA. It was difficult totest the inactivation process on the higher concentrations of the RNasemixture in the presence of RNA, because the RNA was being degraded bythe high concentrations of RNase before the sample could be subjected tothe heating step.

EXAMPLE 4 RNase Inactivation is Dependent on DTT Concentration andTemperature

1. Inactivation of the RNase Mixture

To determine the effective temperature and DTT concentration forribonuclease inactivation, RNase mixtures were prepared to finalconcentrations of 954, 191, 95, and 64 ng/ml. DTT was added to finalconcentrations of 5, 10, and 20 mM. The solutions were incubated at 37°C. or 60° C. for 20 minutes. RNase activity was tested using thenon-isotopic assay. Higher DTT concentrations and higher temperatureswere most effective at inactivating the RNase mixture (Table 1). TheRNase mixture at 954 ng/ml could be inactivated by incubating in 20 mMDTT at 60° C. for 20 minutes. Even an RNase mixture at 64 ng/ml could beinactivated by incubating at 37° C. for 20 minutes with 10 mM DTT. Theinactivation treatments were equally effective in water, in buffer A [1mM sodium citrate (pH 6.4)], in buffer B [50 mM Tris-HCl (pH 7.0)] or inbuffer C [30 mM NaCl: 3 mM sodium citrate (pH 7.5)].

TABLE 1 RNase Inactivation is Dependent on the DTT Concentration and theTemperature in Water or Different Buffers* RNase Mix DTT RNaseInactivation Temp. (ng/ml) (mM) Water Buffer A Buffer B Buffer C (° C.)954 5 − − − − 60 191 5 − − − − 60 95 5 − − − − 60 64 5 + + + + 60 954 10− − − − 37 191 10 − − − − 37 95 10 − − − − 37 64 10 + + + + 37 954 10 −− − − 60 191 10 + + + + 60 95 10 + + + + 60 64 10 + + + + 60 95420 + + + + 60 191 20 + + + + 60 * 1 mM sodium citrate (pH 6.4), BufferA; 50 mM Tris-HCl pH 7.0), Buffer B; 30 mM NaCl; 3 mM sodium citrate pH7.5), Buffer C. (+) indicates that the RNase mixture was inactivated and(−) indicates that the RNase mixture was not activated as determined bythe non-isotopic assay. The RNase cocktail was made to variousconcentrations in different buffers in different DTT concentrations andincubated at 37° C. or 60° C. for 20 minutes. Total mouse RNA was addedto the treated RNase mixture and incubated at 37° C. for one hour,fractionated in a formaldehyde denaturing 1% agarose gel and the RNAvisualized by ethidium bromide staining and UV fluorescence.

2. Heating is Required for Ribonuclease Inactivation

RNase A was diluted to 154 ng/ml in 1 mM sodium citrate (pH 6.4) or inBoshes' (1970) buffer A. DTT was added to the samples to 4 mM or 20 mMand then incubated at 4° C. or 60° C. for 20 minutes. The RNase A wasnot inactivated by 4 mM or 20 mM DTT at 4° C. for 20 minutes. The mousetotal RNA was completely degraded. However, the ribonuclease activitywas completely inactivated with 4 mM or 20 mM DTT coupled with heat at60° C. for 20 minutes. Therefore, the heating step was important forinactivation in this study.

EXAMPLE 5 DTT, DTE and β-mercaptoethanol do not Inhibit EnzymaticReactions in the Presence of RNA

1. DTT, DTE and β-mercaptoethanol do not Inhibit In Vitro Transcriptionor Reverse Transcription Reactions.

DTT, DTE and β-mercaptoethanol at 20 mM in standard in vitrotranscription reactions or standard reverse transcription (RT) reactionsdo not inhibit the efficiency of either of these reactions.

The reverse transcription reactions were performed using theRETROscript™ kit (Ambion, Inc.). The following were assembled with orwithout 20 mM of the reducing reagents in a final volume of 19 μl: dNTPs(0.5 mM each), oligo dT (5 μM), 0.4 μl dCTP[α-³²P] (3000 Ci/mmol), 10 mMTris-HCl (pH 8.3): 50 mM KCl: 1.5 mM MgCl₂, mouse brain total RNA (4μg). The reaction mixtures were incubated at 65° C. for 10 minutes. 1 μl(100 units) of AMV or MMLV reverse transcriptase were added and then thereactions were incubated at 42° C. for 1 hour. Reverse transcription byeither enzyme was not inhibited by DTT (FIG. 2) or by DTE orβ-mercaptoethanol.

The transcription reactions were based on the MAXIscript™ transcriptionkit (Ambion, Inc.). The following were assembled with or without 20 mMreducing reagent in a final volume of 18 μl: NTPs (0.5 mM each), 1 μglinearized DNA template, 2 μl 10×transcription buffer, and 0.2 μl UTP[α-³²P] (800 Ci/mmol). The reaction mixtures were incubated 60° C. for10 minutes and then 2 μl of the either SP6, T7 or T3 RNA polymerase (10units/μl) were added to the reactions, incubated 30° C. for 60 minutesand then TCA precipitated to assess the incorporation of the radioactivelabel. None of the three polymerases were inhibited by DTT (FIG. 3) orby DTE or β-mercaptoethanol.

2. Reducing Reagents and Heat Selectively Inactivate RNases but notDNase I.

DNase I is commonly used to degrade contaminating DNA in RNApreparations. To test whether the inactivation method affected DNase Iactivity, DNase I (final concentration of 0.4 units/μl) was added towater containing 20 mM DTT, DTE or β-mercaptoethanol. The mixture wasincubated at 60° C. for 20 minutes. After the heat treatment, the DNaseI activity was tested by incubating the treated DNase I with plasmid DNA(0.2 μg/μl) at 37° C. for one hour followed by electrophoresis in a 1%agarose gel. The DNA was completely degraded demonstrating that theactivity of the treated DNase I was not affected by the reducing/heattreatment.

EXAMPLE 6 RNase Inactivation is Time Dependent

The RNase mixture (200 ng/ml) was prepared in 1 mM sodium citrate (pH6.4) with 20 mM DTT, followed by heating at 60° C. for 10 seconds, 30seconds, 1 minute, 2 minutes, 4 minutes, 8 minutes, 16 minutes and 20minutes. RNase activity was tested using the non-isotopic assay.Incubation for less than 30 seconds did not inactivate the ribonucleasemixture (FIG. 4). Treatment at 60° C. for 1, 2, and 4 minutesinactivated approximately 50%, 80% and 100% of the activity,respectively. Therefore, inactivation of RNase at 200 ng/ml by 20 mM DTTrequires a 60° C. incubation for at least 4 minutes. Inactivation at ahigher temperature such as 60° C. is preferred over 37° C. because theRNase mixture is inactive at the higher temperature. At 37° C., some ofthe RNA may be degraded by the RNase before the RNase is completelyinactivated.

EXAMPLE 7 Sequential Inactivation of RNases by DTT Coupled with Heating

To perform sequential inactivation of RNases, 20 mM DTT was added to 1mM sodium citrate buffer (pH 6.4). The RNase mixture was added to thebuffer to final concentration of 200 ng/ml. Inactivation was carried outby heating the mixture at 60° C. for 20 minutes. To a portion of theheated RNase, additional RNase mixture was added to 200 ng/ml andfollowed by heating at 60° C. for 20 minutes. Three sequential additionsof ribonuclease mixture and inactivation were performed. A negativecontrol was set up for each sequential addition of ribonuclease mixtureto the buffer but without reheating. RNase activity was measurednon-isotopically. Sequentially added RNases (200 ng/ml) subjected toheating at 60° C. were completely inactivated. In contrast, sequentiallyadded RNases to the same buffer which were not subjected to the 60° C.incubation caused complete RNA degradation, demonstrating that heatingis required for RNase inactivation.

The sequential inactivations of the RNase mixture was possible becausethere was no detectable loss of DTT reducing power with each heatingstep, as measured below by the DTNB assay and therefore, a great excessof reduced DTT was still available to effect the inactivation ofadditional RNase.

To assess the effect that the ribonuclease mixture has on the oxidationof DTT, the RNase mixture at 1 μg/ml was incubated with DTT at 0.05,0.1, 0.15, 0.2 and 0.25 mM at 60° C. for 20 minutes. 0.1 ml of eachsample was added to 0.8 ml of 0.1 M KPO₄ (pH 7.7) and 0.1 ml DTNB in 0.1KPO4 (pH 7.7). Samples which were not treated with heat were alsosubjected to the DTNB assay for comparison. The DTNB reaction mixtureswere incubated at 21° C. for 10 minutes to allow the yellow color todevelop. Absorbances were measured at 412 nm. The results were thatthere was no difference in the reactivity of the DTT whether it had beenused to inactivate the RNase mixture or not. The results showed thatthere was no difference between the DTT used to inactivate the RNasemixture and non-reacted DTT.

EXAMPLE 8 DTT/Heat Inactivation of Ribonucleases is Stable

The RNase mixture was added to a final concentration of 200 ng/ml with20 mM DTT to different solutions [water, TE (pH 8.0), 20×SSC, 10 ×MOPS,1 M Tris (pH 8.0), 1 M Tris (pH 7.0), 1 mM sodium citrate (pH 6.4), 0.5M EDTA (pH 8.0), 5×transcription buffer, 10×transcription buffer,1×blocking buffer, 10×reverse transcription buffer, DNA/RNAhybridization buffer, in situ hybridization buffer, 1 M MgCl₂, 3 Msodium acetate, and formalin tissue fixative] followed by heating at 60°C. for 20 minutes to inactivate RNases. RNase activity was measurednon-isotopically and isotopically.

The DTT-heating procedure inactivated RNase activity in all of buffersand solutions tested. Solutions containing the inactivated RNases werestored at 4° C. for one month and the RNase activity was then retested.Even after one month, none of the solutions had detectable RNaseactivity, indicating that the inactivation procedure is stable. Most ofthe solutions which were not treated with DTT and heat still haddetectable RNase activity.

EXAMPLE 9 Oxidized DTT cannot Inactivate the RNase Mixture

DTT was made to 100 mM in water and incubated at 65° C. for 6 days. Thismaterial was diluted to 50, 100, 150, 200, 250 micromolar in water andthen assayed for its reducing strength using the DTNB test described inExample 7 and compared to the same concentrations of DTT made fresh inwater. The absorbance at 412 nm directly correlates with reducingstrength. The greater the absorbance, the greater the reducing power.The DTT incubated at 65° C. was completely oxidized (FIG. 5).

The DTT incubated at 65° C. for 6 days was then tested for its abilityto inactivate the RNase mixture. The heated DTT was diluted to 20 mM inthe presence of the RNase mixture at 200 ng/ml in water and incubated at60° C. for 20 minutes. RNase activity was measured using thenon-isotopic assay. The oxidized DTT did not inactivate the RNasemixture.

EXAMPLE 10 Inactivation of an Unknown RNase in Alkaline PhosphataseStorage Solution

A commercial solution of unknown composition to the inventors designedto stabilize the storage of alkaline phosphatase was detected to haveRNase activity using the isotopic assay. DTT was added to the alkalinephosphatase storage solution to final concentrations of 0, 2, 5, 10 and20 mM and then incubated at 60° C. for 20 minutes. In the presence of 10and 20 mM DTT, the alkaline phosphatase storage solution developed aprecipitate and was not tested for. RNase activity. The alkalinephosphatase storage solution treated with 0, 2 and 5 mM DTT anduntreated alkaline phosphatase storage solution were tested for RNaseactivity using the isotopic assay. The alkaline phosphatase storagesolution incubated at 60° C. in the presence of 2 and 5 mM DTT had nodetectable RNase activity while the other two samples had similar levelsof RNase activity. Thus, incubating the alkaline phosphatase storagesolution at 60° C. without DTT was not sufficient to inactivate theunknown RNase. This experiment demonstrates that the RNase inactivationprocedure is effective on unknown RNase contaminants.

EXAMPLE 11 Inactivation of an Unknown RNase in Psoralen Biotin

The Millennium™ Markers (Ambion, Inc.) are a set of RNA size markersfrom 0.5 to 9 kb in length. They are used for determining the length ofan unknown RNA. These markers were being labeled with biotin using apsoralen biotin labeling procedure so that they could be detectednon-isotopically on a nylon membrane. However, if the Millennium™Markers were incubated at 37° C. for 16 hours after labeling with thepsoralen biotin, they were degraded. The psoralen biotin wascontaminated with an unknown RNase since the unlabeled Millennium™Markers remained intact after the 37° C., 16 hour incubation. However,if the psoralen biotin labeled Millennium™ Markers were incubated at 90°C. for 10 minutes in the presence of 5 mM DTT after the labelingprocedure to inactivate the RNase activity, then the labeled Millennium™Markers remained intact after incubation at 37° C. for 16 hours.

EXAMPLE 12 Inactivation of Ribonucleases from a Crude Cellular Extractto Protect the Full Length Cellular RNA from Degradation

The reduction-heating process may be used to generate a crude RNase freepreparation of total RNA or polyA+ RNA from cells grown in tissueculture suitable for several different RNA analyses.

Cells grown in tissue culture are pelleted in a microfuge tube and thegrowth medium is removed. The cells are placed on ice. An isotonicsolution such as phosphate buffered saline (PBS) containing 20 mM DTT isadded to the cells to resuspend them. The cell suspension is quicklyheated to between 60° C. and 90° C. for 10 to 20 minutes. The heatingstep would have two functions: 1) It denatures the cell membrane,releasing the cells' RNA and other contents into the PBS, and 2) Itinactivates the RNases, thus preventing the degradation of the RNA.After heating, the cell suspension is centrifuged at high speed topellet insoluble debris.

DNase I may be added to the cell suspension after the heat inactivationof the RNases. The contents would be incubated at 37° C. for 15 minutesto digest the cellular DNA. Finally, EDTA is added to 2 mM and contentsincubated at 65° C. for 10 minutes to inactivate the DNase I. The cellsuspension should now be suitable for RT-PCR.

It may be necessary to perform the initial heat inactivation at a highertemperature to achieve minimal RNA degradation. This process may also beperformed on prokaryotic cells. It will be important to have a fast ramptime for the initial heat inactivation to minimize the time during whichthe temperature will be optimal for RNase activity.

EXAMPLE 13 Cysteine can be used to Heat Inactivate the RNase Mixture.

The RNase mixture was made to 200 ng/ml in 1 mM sodium citrate (pH 6.4)at 10, 20, 40 and 60 mM cysteine. The samples were incubated at 68° C.for 20 minutes. Total mouse RNA substrate was added to the samples andincubated for 37° C. for 1 hour, fractionated on a formaldehyde 1%agarose gel and detected by UV fluorescence. All of the RNase samplestreated with cysteine and heat were inactivated.

EXAMPLE 14 DTT and Heat can be used to Enhance to Preservative Activityof RNAlater™

The term “RNAlater™” is a trademark of Ambion, Inc., for certaincommercial formulations of the RNA preservation media. This technologyis described in detail in U.S. Pat. application Ser. No. 09/127,435,filed Jul. 31, 1998, now U.S. Pat. No. 6,204,375, entitled “METHODS ANDREAGENTS FOR PRESERVING RNA IN CELL AND TISSUE SAMPLES,” which isincorporated herein by reference. This reagent functions by rapidlyinfiltrating cells with a high concentration of ammonium sulfate,causing a mass precipitation of cellular proteins. Importantly, cellularstructure remains intact. The advantage of this is that cells can bepreserved and still identified histologically. RNAlater™ inhibits RNaseactivity in tissues and thereby maintain full length RNA. Sample tissuesare dropped in RNAlater™ solution. The RNA will remain intact for 1 dayif kept at 37° C., intact for 1 week if kept at 21° C., and indefinitelyat 4° C. The RNA may be preserved for longer periods at the highertemperatures if the ribonucleases in the tissues were more fullyinactivated.

A reducing agent such as DTT or cysteine may be added to the RNAlater™solution to 20 mM. When a tissue is added to the RNAlater™/DTT solutionit would be heated to 40 to 600° C. for 10 to 20 minutes, therebyinactivating the RNases by reduction. In this manner, the advantages ofboth solutions may be realized with the result being even betterassurance of protection from RNases.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of treating a sample to prepare anadmixture with no detectable RNase A, RNase 1, and/or RNase T1 activitycomprising: (a) obtaining a sample; (b) obtaining a thiol-containingreducing agent; (c) admixing the sample and the thiol-containingreducing agent to prepare an admixture; and (d) heating the admixture toa temperature and for a time required to result in the inactivation ofany of RNase A, RNase 1, and/or RNase T1 present in the admixture;wherein there is no detectable RNase A, RNase 1, or RNase T1 activity inthe admixture after the heating step.
 2. The method of claim 1, furthercomprising treating a sample containing detectable RNase A, RNase 1,and/or RNase T1 activity prior to treatment.
 3. The method of claim 2,wherein the detectable activity is RNase A activity.
 4. The method ofclaim 2, wherein the detectable activity is RNase 1 activity.
 5. Themethod of claim 2, wherein the detectable activity is RNase T1 activity.6. The method of claim 1, further comprising treating a samplecontaining detectable RNase A, RNase 1, and RNase T1 activity prior totreatment.
 7. The method of claim 1, wherein said thiol-based reducingagent is dithiothreitol (DTT), β-mercaptoethanol, dithioerythritol(DTE), or cysteine.
 8. The method of claim 1, wherein the finalconcentration of the thiol-based reducing agent is between 1 and 200 mMin the admixture.
 9. The method of claim 7, wherein said reducing agentis dithiothreitol (DTT).
 10. The method of claim 9, wherein the finalconcentration of dithiothreitol (DTT) is between 1 and 200 mM in theadmixture.
 11. The method of claim 7, wherein said reducing agent isβ-mercaptoethanol.
 12. The method of claim 11, wherein the finalconcentration of β-mercaptoethanol is between 1 and 200 mM in theadmixture.
 13. The method of claim 7, wherein said reducing agent iscysteine.
 14. The method of claim 13, wherein the final concentration ofcysteine is between 1 and 200 mM in the admixture.
 15. The method ofclaim 1, further comprising mixing the thiol-based reducing agent with abuffer prior to admixing in step (c).
 16. The method of claim 15,wherein said buffer further comprises a chelator.
 17. The method ofclaim 16, wherein the chelator is sodium citrate,ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), orethylenediamine tetraacetic acid (EDTA).
 18. The method of claim 1,wherein the admixture is heated to at least 37° C.
 19. The method ofclaim 1, wherein the admixture is heated to at least 60° C.
 20. Themethod of claim 1, wherein the mixture is heated for at least fourminutes.
 21. The method of claim 1, wherein the sample contains RNA. 22.The method of claim 21, wherein the sample contains purified RNA. 23.The method of claim 21, wherein the sample is a crude cell preparation.24. The method of claim 21, wherein the sample is a cell sample.
 25. Themethod of claim 21, further comprising performing a reversetranscriptase-polymerase chain reaction procedure with the admixtureafter the heating step.
 26. The method of claim 1, wherein the samplecomprises water, or further comprises Tris/EDTA (TE) buffer, NaCl/sodiumcitrate buffer (SSC), MOPS/sodium acetate/EDTA buffer (MOPS), Trisbuffer, aqueous ethylenediamine tetraacetic acid (EDTA), nucleic acidhybridization buffer, sodium acetate buffer, formalin tissue fixativesolution, in situ hybridization buffer, or nucleic acid storagebuffer/solution.
 27. The method of claim 1, further comprisingconducting a second heating of the admixture for a temperature and for atime wherein there is no detectable RNase A, RNase 1, and/or RNase T1activity present in the admixture after the second heating.
 28. A methodof treating an RNA sample to prepare an RNA-containing admixture with nodetectable RNase A, RNase 1, and/or RNase T1 activity comprising: (a)obtaining an RNA sample; (b) obtaining a thiol-containing reducingagent; (c) admixing the sample and the thiol-containing reducing agentto prepare an admixture; and (d) heating the admixture to a temperatureand for a time required to result in the inactivation of any of RNase A,RNase 1, and/or RNase T1 present in the admixture; wherein there is nodetectable RNase A, RNase 1, or RNase T1 activity in the admixture afterthe heating step.
 29. The method of claim 28, wherein the samplecontains purified RNA.
 30. The method of claim 28, wherein the sample isa crude cell preparation.
 31. The method of claim 28, wherein the sampleis a cell sample.
 32. The method of claim 28, further comprisingperforming a reverse transcriptase-polymerase chain reaction procedurewith the admixture after the heating step.
 33. The method of claim 28,wherein said thiol-based reducing agent is dithiothreitol (DTT),β-mercaptoethanol, dithioerythritol DTE), or cysteine.
 34. The method ofclaim 28, wherein the final concentration of the thiol-based reducingagent is between 1 and 200 mM in the admixture.
 35. The method of claim28, wherein the admixture is heated to at least 37° C.
 36. The method ofclaim 28, wherein the admixture is heated at least 60° C.
 37. The methodof claim 28, wherein the mixture is heated for at least four minutes.38. The method of claim 28, wherein said RNA sample contains detectableRNase A, RNase 1, and/or RNase T1 activity prior to treatment.